Alternative mechanisms of initiating translation of mammalian mRNAs

2005 ◽  
Vol 33 (6) ◽  
pp. 1231-1241 ◽  
Author(s):  
R.J. Jackson
Keyword(s):  
Site Selection ◽  
Mammalian Cells ◽  
Ribosomal Subunit ◽  
Aggregate Size ◽  
Mrna Translation ◽  
Initiation Site ◽  
Initiation Factor ◽  
Viral Mrnas ◽  
Initiation Factors ◽  
Scanning Mechanism

Of all the steps in mRNA translation, initiation is the one that differs most radically between prokaryotes and eukaryotes. Not only is there no equivalent of the prokaryotic Shine–Dalgarno rRNA–mRNA interaction, but also what requires only three initiation factor proteins (aggregate size ∼125 kDa) in eubacteria needs at least 28 different polypeptides (aggregate >1600 kDa) in mammalian cells, which is actually larger than the size of the 40 S ribosomal subunit. Translation of the overwhelming majority of mammalian mRNAs occurs by a scanning mechanism, in which the 40 S ribosomal subunit, primed for initiation by the binding of several initiation factors including the eIF2 (eukaryotic initiation factor 2)–GTP–MettRNAi complex, is loaded on the mRNA immediately downstream of the 5′-cap, and then scans the RNA in the 5′→3′ direction. On recognition of (usually) the first AUG triplet via base-pairing with the Met-tRNAi anticodon, scanning ceases, triggering GTP hydrolysis and release of eIF2–GDP. Finally, ribosomal subunit joining and the release of the other initiation factors completes the initiation process. This sketchy outline conceals the fact that the exact mechanism of scanning and the precise roles of the initiation factors remain enigmatic. However, the factor requirements for initiation site selection on some viral IRESs (internal ribosome entry sites/segments) are simpler, and investigations into these IRES-dependent mechanisms (particularly picornavirus, hepatitis C virus and insect dicistrovirus IRESs) have significantly enhanced our understanding of the standard scanning mechanism. This article surveys the various alternative mechanisms of initiation site selection on mammalian (and other eukaryotic) cellular and viral mRNAs, starting from the simplest (in terms of initiation factor requirements) and working towards the most complex, which paradoxically happens to be the reverse order of their discovery.

scholarly journals Assembly of 48S Translation Initiation Complexesfrom Purified Components with mRNAs That Have Some Base Pairingwithin Their 5′ UntranslatedRegions

2003 ◽  
Vol 23 (24) ◽  
pp. 8925-8933 ◽  
Author(s):  
Sergei E. Dmitriev ◽  
Ilya M. Terenin ◽  
Yan E. Dunaevsky ◽  
William C. Merrick ◽  
Ivan N. Shatsky
Keyword(s):  
Translation Initiation ◽  
Mammalian Cells ◽  
Ribosomal Subunit ◽  
Initiation Factor ◽  
Beta Globin ◽  
Globin Mrna ◽  
Initiation Factors ◽  
40S Ribosomal Subunit ◽  
Cellular Mrnas ◽  
Actin Mrna

ABSTRACT The reconstitution of translation initiation complexes from purified components is a reliable approach to determine the complete set of essential canonical initiation factors and auxiliary proteins required for the 40S ribosomal subunit to locate the initiation codon on individual mRNAs. Until now, it has been successful mostly for formation of 48S translation initiation complexes with viral IRES elements. Among cap-dependent mRNAs, only globin mRNAs and transcripts with artificial 5′ leaders were amenable to this assembly. Here, with modified conditions for the reconstitution, 48S complexes have been successfully assembled with the 5′ UTR of beta-actin mRNA (84 nucleotides) and the tripartite leader of adenovirus RNAs (232 nucleotides), though the latter has been able to use only the scanning rather then the shunting model of translation initiation with canonical initiation factors. We show that initiation factor 4B is essential for mRNAs that have even a rather moderate base pairing within their 5′ UTRs (with the cumulative stability of the secondary structure within the entire 5′ UTR < −13 kcal/mol) and not essential for beta-globin mRNA. A recombinant eIF4B poorly substitutes for the native factor. The 5′ UTRs with base-paired G residues reveal a very sharp dependence on the eIF4B concentration to form the 48S complex. The data suggest that even small variations in concentration or activity of eIF4B in mammalian cells may differentially affect the translation of different classes of cap-dependent cellular mRNAs.

scholarly journals Selective Modification of Eukaryotic Initiation Factor 4F (eIF4F) at the Onset of Cell Differentiation: Recruitment of eIF4GII and Long-Lasting Phosphorylation of eIF4E

2004 ◽  
Vol 24 (11) ◽  
pp. 4920-4928 ◽  
Author(s):  
Sandrine Caron ◽  
Martine Charon ◽  
Elisabeth Cramer ◽  
Nahum Sonenberg ◽  
Isabelle Dusanter-Fourt
Keyword(s):  
Functional Analysis ◽  
Cell Differentiation ◽  
Mammalian Cells ◽  
Ribosomal Subunit ◽  
Mrna Translation ◽  
Scaffold Protein ◽  
Synergistic Action ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Critical Link

ABSTRACT mRNA translation is mainly regulated at the level of initiation, a process that involves the synergistic action of the 5′ cap structure and the 3′ poly(A) tail at the ends of eukaryotic mRNA. The eukaryote initiation factor 4G(eIF4G) is a pivotal scaffold protein that forms a critical link between mRNA cap structure, poly(A) tail, and the small ribosomal subunit. There are two functional homologs of eIF4G in mammals, the original eIF4G, renamed eIF4GI, and eIF4GII that functionally complements eIF4GI. To date, biochemical and functional analysis have not identified differential activities for eIF4GI and eIF4GII. In this report, we demonstrate that eIF4GII, but not eIF4GI, is selectively recruited to capped mRNA at the onset of cell differentiation. This recruitment is coincident with a strong and long-lasting phosphorylation of eIF4E and the release of 4E-BP1, a suppressor of eIF4E function, from the cap structure, without a concomitant change in 4E-BP1's phosphorylation. Our data further indicate that cytokines such as thrombopoietin can differentially regulate eIF4GI/II activities. These results provide the first evidence that eIF4GI/II does fulfill selective roles in mammalian cells.

Hormone-induced meiotic maturation in Xenopus oocytes occurs independently of p70s6k activation and is associated with enhanced initiation factor (eIF)-4F phosphorylation and complex formation

1995 ◽  
Vol 108 (4) ◽  
pp. 1751-1760 ◽  
Author(s):  
S.J. Morley ◽  
V.M. Pain
Keyword(s):  
Complex Formation ◽  
Mammalian Cells ◽  
De Novo ◽  
Ribosomal Subunit ◽  
Early Time ◽  
Initiation Factor ◽  
Meiotic Maturation ◽  
Initiation Factors ◽  
Ribosomal Protein S6 ◽  
Time Critical

Hormone-induced meiotic maturation of the Xenopus oocyte is regulated by complex changes in protein phosphorylation. It is accompanied by a stimulation in the rate of translation, manifest at the level of polypeptide chain initiation. At laser times in the maturation process, this reflects an increased ability for mRNA to interact with the 40 S ribosomal subunit. In mammalian cells there is growing evidence for the regulation of translation by phosphorylation of ribosomal protein S6 and of initiation factors responsible for the binding of mRNA to ribosomes. In this report, we show that although the 70 kDa form of S6 kinase is activated within 1.5 hours in response to progesterone or insulin, a time critical for protein synthesis, its activation is not required for hormone-induced stimulation of translation rates or maturation. In response to progesterone, activation of translation occurs in parallel with enhanced phosphate labelling of eIF-4 alpha and eIF-4 gamma and eIF-4F complex formation, events which are thought to facilitate the interaction of eIF-4F with the mRNA cap structure. However, with insulin, activation of translation occurs prior to detectable de novo phosphorylation of eIF-4F, although a small enhancement of turnover of phosphate on eIF-4 alpha may occur at this early time. With either hormone, enhanced phosphate labelling of eIF-4 alpha is shown to reflect activation of eIF-4 alpha kinase(s), which coincides temporally with activation of p42 MAP and p90rsk kinases. The possible role of initiation factor modification on increased translation rates during meiotic maturation is discussed.

BIOCHEMICAL MECHANISM TO CONTROL PROTEIN SYNTHESIS mRNA SPECIFIC INITIATION FACTORS

1973 ◽  
Vol 74 (Suppl) ◽  
pp. S54-S74 ◽  
Author(s):  
M. Revel ◽  
Y. Groner ◽  
Y. Pollack ◽  
D. Cnaani ◽  
H. Zeller ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Mammalian Cells ◽  
Mrna Translation ◽  
Initiation Factor ◽  
Interference Factor ◽  
E Coli ◽  
Initiation Factors ◽  
Virus Rna ◽  
Globin Synthesis

ABSTRACT Studies with cell fre systems of protein synthesis have shown that mRNAs are not translated uniformly by ribosomes. In particular the existence of mRNA specific initiation factors which stimulate or inhibit the translation of individual cistrons is now well recognized. We originally reported that E. coli initiation factor IF3 activity can be separated into subfractions differing in their activity toward MS2 and T4 mRNA. Our recent work, however, indicates that the specificity of IF3 for mRNAs actually results from its combination with "interference" protein factors. These proteins act in the presence of IF3 to stimulate the translation of certain cistrons while inhibiting that of others. We have isolated three interference factors from E. coli and characterized some of their cistron specificities on MS2 RNA, T4 and T7 early and late mRNAs. These factors can explain the heterogeneity of IF3 toward various mRNA. In vivo variations in the activity of ribosomes can result from changes either in IF3 or in an interference factor. We have also recently purified a mRNA-specific initiation factor from mammalian cells. This factor isolated from the ribosomal wash of rabbit reticulocytes stimulates haemoglobin mRNA translation but not that of Mengo virus RNA, in a crude extract from Krebs ascites cells. This factor stimulates a globin synthesis. Ribosome activity may be, therefore, regulated both quantitatively and qualitatively by such factors.

Multiple isoforms of the translation initiation factor eIF4GII are generated via use of alternative promoters, splice sites and a non-canonical initiation codon

2012 ◽  
Vol 448 (1) ◽  
pp. 1-11 ◽  
Author(s):  
Mark J. Coldwell ◽  
Ulrike Sack ◽  
Joanne L. Cowan ◽  
Rachel M. Barrett ◽  
Markete Vlasak ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Ribosomal Subunit ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Initiation Codon ◽  
Alternative Promoters ◽  
Multiple Promoters ◽  
N Terminus ◽  
Eukaryotic Initiation Factor 4G

During the initiation stage of eukaryotic mRNA translation, the eIF4G (eukaryotic initiation factor 4G) proteins act as an aggregation point for recruiting the small ribosomal subunit to an mRNA. We previously used RNAi (RNA interference) to reduce expression of endogenous eIF4GI proteins, resulting in reduced protein synthesis rates and alterations in the morphology of cells. Expression of EIF4G1 cDNAs, encoding different isoforms (f–a) which arise through selection of alternative initiation codons, rescued translation to different extents. Furthermore, overexpression of the eIF4GII paralogue in the eIF4GI-knockdown background was unable to restore translation to the same extent as eIF4GIf/e isoforms, suggesting that translation events governed by this protein are different. In the present study we show that multiple isoforms of eIF4GII exist in mammalian cells, arising from multiple promoters and alternative splicing events, and have identified a non-canonical CUG initiation codon which extends the eIF4GII N-terminus. We further show that the rescue of translation in eIF4GI/eIF4GII double-knockdown cells by our novel isoforms of eIF4GII is as robust as that observed with either eIF4GIf or eIF4GIe, and more than that observed with the original eIF4GII. As the novel eIF4GII sequence diverges from eIF4GI, these data suggest that the eIF4GII N-terminus plays an alternative role in initiation factor assembly.

4E-BP1 and S6K1: translational integration sites for nutritional and hormonal information in muscle

2000 ◽  
Vol 279 (4) ◽  
pp. E715-E729 ◽  
Author(s):  
O. Jameel Shah ◽  
Joshua C. Anthony ◽  
Scot R. Kimball ◽  
Leonard S. Jefferson
Keyword(s):  
Translational Control ◽  
Mrna Translation ◽  
Cellular Protein ◽  
Initiation Factor ◽  
Translational Repressor ◽  
Initiation Factors ◽  
Initiation Phase ◽  
Eukaryotic Initiation Factor 4E ◽  
Integration Sites ◽  
A Site

Maintenance of cellular protein stores in skeletal muscle depends on a tightly regulated synthesis-degradation equilibrium that is conditionally modulated under an extensive range of physiological and pathophysiological circumstances. Recent studies have established the initiation phase of mRNA translation as a pivotal site of regulation for global rates of protein synthesis, as well as a site through which the synthesis of specific proteins is controlled. The protein synthetic pathway is exquisitely sensitive to the availability of hormones and nutrients and employs a comprehensive integrative strategy to interpret the information provided by hormonal and nutritional cues. The translational repressor, eukaryotic initiation factor 4E binding protein 1 (4E-BP1), and the 70-kDa ribosomal protein S6 kinase (S6K1) have emerged as important components of this strategy, and together they coordinate the behavior of both eukaryotic initiation factors and the ribosome. This review discusses the role of 4E-BP1 and S6K1 in translational control and outlines the mechanisms through which hormones and nutrients effect changes in mRNA translation through the influence of these translational effectors.

scholarly journals A Complementary Mechanism of Bacterial mRNA Translation Inhibition by Tetracyclines

2021 ◽  
Vol 12 ◽  
Author(s):  
Victor Barrenechea ◽  
Maryhory Vargas-Reyes ◽  
Miguel Quiliano ◽  
Pohl Milón
Keyword(s):  
Protein Synthesis ◽  
Translation Initiation ◽  
Ribosomal Subunit ◽  
Mrna Translation ◽  
Resistance Mechanisms ◽  
Dissociation Rate ◽  
Initiation Factor ◽  
Translation Elongation ◽  
Initiation Phase ◽  
Complementary Mechanism

Tetracycline has positively impacted human health as well as the farming and animal industries. Its extensive usage and versatility led to the spread of resistance mechanisms followed by the development of new variants of the antibiotic. Tetracyclines inhibit bacterial growth by impeding the binding of elongator tRNAs to the ribosome. However, a small number of reports indicated that Tetracyclines could also inhibit translation initiation, yet the molecular mechanism remained unknown. Here, we use biochemical and computational methods to study how Oxytetracycline (Otc), Demeclocycline (Dem), and Tigecycline (Tig) affect the translation initiation phase of protein synthesis. Our results show that all three Tetracyclines induce Initiation Factor IF3 to adopt a compact conformation on the 30S ribosomal subunit, similar to that induced by Initiation Factor IF1. This compaction was faster for Tig than Dem or Otc. Furthermore, all three tested tetracyclines affected IF1-bound 30S complexes. The dissociation rate constant of IF1 in early 30S complexes was 14-fold slower for Tig than Dem or Otc. Late 30S initiation complexes (30S pre-IC or IC) exhibited greater IF1 stabilization by Tig than for Dem and Otc. Tig and Otc delayed 50S joining to 30S initiation complexes (30S ICs). Remarkably, the presence of Tig considerably slowed the progression to translation elongation and retained IF1 in the resulting 70S initiation complex (70S IC). Molecular modeling of Tetracyclines bound to the 30S pre-IC and 30S IC indicated that the antibiotics binding site topography fluctuates along the initiation pathway. Mainly, 30S complexes show potential contacts between Dem or Tig with IF1, providing a structural rationale for the enhanced affinity of the antibiotics in the presence of the factor. Altogether, our data indicate that Tetracyclines inhibit translation initiation by allosterically perturbing the IF3 layout on the 30S, retaining IF1 during 70S IC formation, and slowing the transition toward translation elongation. Thus, this study describes a new complementary mechanism by which Tetracyclines may inhibit bacterial protein synthesis.

scholarly journals Measles Virus N Protein Inhibits Host Translation by Binding to eIF3-p40

2007 ◽  
Vol 81 (21) ◽  
pp. 11569-11576 ◽  
Author(s):  
Hiroki Sato ◽  
Munemitsu Masuda ◽  
Moeko Kanai ◽  
Kyoko Tsukiyama-Kohara ◽  
Misako Yoneda ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Mammalian Cells ◽  
Measles Virus ◽  
Encephalomyocarditis Virus ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Translation System ◽  
Dependent Manner ◽  
Initiation Factors ◽  
Human T Cell

ABSTRACT The nonsegmented, negative-sense RNA genome of measles virus (MV) is encapsidated by the virus-encoded nucleocapsid protein (N). In this study, we searched for N-binding cellular proteins by using MV-N as bait and screening the human T-cell cDNA library by yeast two-hybrid assay and isolated the p40 subunit of eukaryotic initiation factor 3 (eIF3-p40) as a binding partner. The interaction between MV-N and eIF3-p40 in mammalian cells was confirmed by coimmunoprecipitation. Since eIF3-p40 is a translation initiation factor, we analyzed the potential inhibitory effect of MV-N on protein synthesis. Glutathione S-transferase (GST)-fused MV-N (GST-N) inhibited translation of reporter mRNAs in rabbit reticulocyte lysate translation system in a dose-dependent manner. Encephalomyocarditis virus internal ribosomal entry site-mediated translation, which requires canonical initiation factors to initiate translation, was also inhibited by GST-N. In contrast, a unique form of translation mediated by the intergenic region of Plautia stali intestine virus, which can assemble 80S ribosomes in the absence of canonical initiation factors, was scarcely affected by GST-N. In vivo expression of MV-N induced by the Cre/loxP switching system inhibited the synthesis of a transfected reporter protein, as well as overall protein synthesis. These results suggest that MV-N targets eIF3-p40 and may be involved in inhibiting MV-induced host translation.

scholarly journals A Retrospective on eIF2A—and Not the Alpha Subunit of eIF2

2020 ◽  
Vol 21 (6) ◽  
pp. 2054
Author(s):  
Anton A. Komar ◽  
William C. Merrick
Keyword(s):  
Translational Control ◽  
Ribosomal Subunit ◽  
Initiation Factor ◽  
Single Chain ◽  
Initiation Factors ◽  
Eif2α Phosphorylation ◽  
Specific Mrnas ◽  
Internal Initiation ◽  
And Function ◽  
Polypeptide Chains

Initiation of protein synthesis in eukaryotes is a complex process requiring more than 12 different initiation factors, comprising over 30 polypeptide chains. The functions of many of these factors have been established in great detail; however, the precise role of some of them and their mechanism of action is still not well understood. Eukaryotic initiation factor 2A (eIF2A) is a single chain 65 kDa protein that was initially believed to serve as the functional homologue of prokaryotic IF2, since eIF2A and IF2 catalyze biochemically similar reactions, i.e., they stimulate initiator Met-tRNAi binding to the small ribosomal subunit. However, subsequent identification of a heterotrimeric 126 kDa factor, eIF2 (α,β,γ) showed that this factor, and not eIF2A, was primarily responsible for the binding of Met-tRNAi to 40S subunit in eukaryotes. It was found however, that eIF2A can promote recruitment of Met-tRNAi to 40S/mRNA complexes under conditions of inhibition of eIF2 activity (eIF2α-phosphorylation), or its absence. eIF2A does not function in major steps in the initiation process, but is suggested to act at some minor/alternative initiation events such as re-initiation, internal initiation, or non-AUG initiation, important for translational control of specific mRNAs. This review summarizes our current understanding of the eIF2A structure and function.

scholarly journals PABP1 and eIF4GI associate with influenza virus NS1 protein in viral mRNA translation initiation complexes

2003 ◽  
Vol 84 (12) ◽  
pp. 3263-3274 ◽  
Author(s):  
Idoia Burgui ◽  
Tomás Aragón ◽  
Juan Ortín ◽  
Amelia Nieto
Keyword(s):  
Influenza Virus ◽  
Translation Initiation ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Published Data ◽  
Ns1 Protein ◽  
Viral Mrnas ◽  
Independent Manner ◽  
Cellular Mrnas

It has previously been shown that influenza virus NS1 protein enhances the translation of viral but not cellular mRNAs. This enhancement occurs by increasing the rate of translation initiation and requires the 5′UTR sequence, common to all viral mRNAs. In agreement with these findings, we show here that viral mRNAs, but not cellular mRNAs, are associated with NS1 during virus infection. We have previously reported that NS1 interacts with the translation initiation factor eIF4GI, next to its poly(A)-binding protein 1 (PABP1)-interacting domain and that NS1 and eIF4GI are associated in influenza virus-infected cells. Here we show that NS1, although capable of binding poly(A), does not compete with PABP1 for association with eIF4GI and, furthermore, that NS1 and PABP1 interact both in vivo and in vitro in an RNA-independent manner. The interaction maps between residues 365 and 535 in PABP1 and between residues 1 and 81 in NS1. These mapping studies, together with those previously reported for NS1–eIF4GI and PABP1–eIF4GI interactions, imply that the binding of all three proteins would be compatible. Collectively, these and previously published data suggest that NS1 interactions with eIF4GI and PABP1, as well as with viral mRNAs, could promote the specific recruitment of 43S complexes to the viral mRNAs.

Sign in / Sign up

Export Citation Format

Share Document